Mage-a3/hpv 16 peptide vaccines for head and neck cancer

ABSTRACT

The present invention relates to Trojan antigens, and immunogenic compositions comprising the Trojan antigens. The present invention also relates to methods of generating an immune response in a subject using the Trojan antigens or immunogenic compositions. The present invention further relates to methods of treating squamous cell carcinoma of the head and neck (SCCHN) using the Trojan antigens and immunogenic compositions of the present invention.

The present application claims benefit of U.S. provisional applicationNo. 60/667,060, filed Apr. 1, 2005, incorporated herein by reference inits entirety.

BACKGROUND OF THE INVENTION

Squamous cell carcinoma of the head and neck (SCCHN) effects 43,000individuals in the United States annually with an estimated 5-yearoverall survival of 50% (R. M. Byers, Dr. Martin: How are we doing in2000? Archives of Otolaryngology-Head and Neck Surgery 127:759-765(2001)). For some patients who develop local or distant metastasesfollowing primary therapy, surgical salvage is a viable therapeuticoption. The remainder of individuals is forced to choose betweenpalliative chemotherapy and supportive care. In order to improve bothsurvival and quality of life for patients with unresectable disease, newtherapeutic alternatives are mandated.

One treatment option is the use of T cell-specific immunotherapy tostimulate a patient's anti-tumor immune response. Several T cell basedstrategies have demonstrated clinical efficacy for the treatment ofunresectable tumors of diverse histologic types (Kugler et al.Regression of human metastatic renal cell carcinoma after vaccinationwith tumor cell=dendritic cell hybrids. Nature Medicine 6(3):332-6(2000); Nestle et al. Vaccination of melanoma patients with peptide- ortumor lysate-pulsed dendritic cells. Nature Medicine 4(3):328-32 (1998);Rosenberg et al. Immunologic and therapeutic evaluation of a syntheticpeptide vaccine for the treatment of patients with metastatic melanoma.Nature Medicine 4(3):321-327 (1998)). However, the majority of trialshave failed to demonstrate any therapeutic benefit (Chang et al. A phaseI trial of tumor lysate-pulsed dendritic cells in the treatment ofadvanced cancer. Clinical Cancer Research 8(4):1021-1032 (2002)). Thecauses of these failures are multi-factorial, but are likely related towell characterized immunologic defects within the target population,including aberrant antigen processing and presentation, which restrict Tcell function. Specifically, tumor bearing patients have demonstrabledefects in antigen presentation, both at the tumor cell and professionalantigen presenting cell (APC) levels, e.g., down regulation of TAP andHLA molecules, which, in some cases, can be overcome by theadministration of interferon (Seliger et al. Antigen-processingmachinery breakdown and tumor growth. Immunology Today 21(9):455-64(2000); Marincola et al. Escape of human solid tumors from T-cellrecognition: Molecular mechanisms and functional significance, AdvancedImmunology 74:181-273 (2000)). Additionally, in cancer patients T cellsare found to be tolerized or improperly activated which might be causedby down regulation of the zeta chain of the T cell receptor (TCR) andP56LCK signaling molecule (Mizoguchi et al. Alterations in signaltransduction molecules in T lymphocytes from tumor-bearing mice. Science258(5089):1795-8 (1992)).

Classic experiments by Gross in the 1940s created the foundation for thefield of tumor immunology, demonstrating that C3H mice capable ofrejecting a syngeneic murine sarcoma developed protective immunity tosubsequent injections of the same tumor, but not to a spontaneouslyarising murine mastocytoma (L. Gross, Intradermal immunization of C3Hmice against a sarcoma that originated in an animal of the same line.Cancer Res. 326-333 (1943)). This immune response is directed againsttumor specific target antigens and is cellular in nature as lymphocytes,but not serum, from previously immunized animals are effective in lysingtumor (Prehn et al. Immunity to methylcolanthrene-induced sarcomas.Journal of the National Cancer Institute 6:769-778 (1957).

It is now clear that tumor antigens are presented in the context ofspecific class I and class II HLA molecules. Class I presentation, inthe presence of appropriate costimulation, is thought to stimulate acytolytic CD8+ T cell response, while antigen presentation in thecontext of class II molecules is postulated to stimulate a CD4+ helper Tcell response (Townsend et al., Antigen recognition by classI-restricted T lymphocytes. Annual Review of Immunology 7:601-624(1989)). Tumors can evade the immune response by manipulating theseantigen presentation pathways. Specifically, direct tumor presentationof antigen in the absence of costimulation results in T cell anergy (D.M. Pardoll, Cancer vaccines. Nature Medicine 4(5 supp):525-531 (1998)).Additionally, down regulation of either HLA molecules on the tumorsurface or tumor antigen expression limits the efficacy ofantigen-specific cytotoxic T cells (Seliger et al. Antigen-processingmachinery breakdown and tumor growth. Immunology Today 21(9):455-64(2000)). Finally, tumors can upregulate non-classical HLA molecules,e.g., HLA G, which are thought to suppress T cell anti-tumor immunity(Rouas-Freiss et al. HLA-G promotes immune tolerance. Journal ofBiological Regulators & Homeostatic Agents 14(2):93-8 (2000)). Thesemolecular methods of protection provide clear evidence that CTLs induceselective pressure on tumors and can potentially be harnessed withtherapeutic intent.

Several strategies have been employed to prime the anti-tumor T cellresponse. A major advance in the field of immunotherapy is the clinicalapplication of a class of professional antigen presenting cells (APC),termed dendritic cells (DC), into clinical trials. Dendritic cells arethought to stimulate the T cell anti-tumor response by the presentationof tumor associated antigens in the context of class I and II MHC,co-stimulatory molecules, and appropriate chemokines/cytokines (Gilboaet al. Immunotherapy of cancer with dendritic-cell-based vaccines.Cancer Immunology, Immunotherapy 46(2):82-7 (1998)). Presentation ofputative tumor antigens by DC is postulated to effectively overcometumor induced tolerance (Dallal et al. The dendritic cell and humancancer vaccines, Current Opinion in Immunology 12(5):583-8 (2000)). Thissuggests that vaccine approaches using DC primed with tumor specificantigens will be an effective means to stimulate tumor specificimmunity.

Recent evidence suggests that DC primed with tumor-associated antigensin the form of peptides, tumor lysates, or tumor RNA are capable ofmediating a potent anti-tumor immune response (Nair et al. Regression oftumors in mice vaccinated with professional antigen-presenting cellspulsed with tumor extracts. International Journal of Cancer 70(6):706-15(1997)). While ex vivo maturation of DC is one method of priming DC,such maturation is expensive and can lack reproducibility. Specifically,because large numbers of DC are required for vaccine preparation, 1-2leukophoreses are often required.

One means to potentially eliminate problems associated with ex vivomaturation is through the use of DC stimulation in vivo. Monocytescultured in appropriate concentrations of IL-4 and GM-CSF transform intoimmature DC (Banchereau et al. Dendritic cells and the control ofimmunity. Nature 392(6673):245-52 (1998); R. M. Steinman, The dendriticcell system and its role in immunogenicity. Annual Review of Immunology9:271-96 (1991)). GM-CSF is the critical component for stimulating DCmaturation, phagocytosis, migration, and HLA class II expression (J. O.Armitage. Emerging applications of recombinant humangranulocyte-macrophage colony-stimulating factor. Blood 92(12):4491-508(1998)). The majority of clinical trials have cultured DC precursors inrecombinant GM-CSF, with subsequent administration back to the host aspart of a tumor vaccine. Recent data from Disis et al. demonstrates thatthe presentation of tumor peptides in a rat model is enhanced bysubcutaneous or intradermal administration of GM-CSF (Disis et al.Granulocyte-macrophage colony-stimulating factor: an effective adjuvantfor protein and peptide-based vaccines. Blood 88(1):202-10 (1996)). Theconcept of in vivo administration of GM-CSF in combination with anautologous vaccine has been clinically evaluated in patients withadvanced melanoma. Specifically, using an autologous melanoma vaccine incombination with GM-CSF and BCG, Leong et al. demonstrated a 10%complete response rate with an equal number of partial responders in acohort of 20 patients (Leong et al. Recombinant human granulocytemacrophage-colony stimulating factor (rhGM-CSF) and autologous melanomavaccine mediate tumor regression in patients with metastatic melanoma.Journal of Immunotherapy 22(2):166-74 (1999)). Using a modification ofthis approach, Soiffer et al. demonstrated that a vaccine composed ofirradiated melanoma cells engineered to secrete GM-CSF, stimulated Tcell mediated tumor destruction in 11/16 patients (Soiffer et al.Vaccination with irradiated autologous melanoma cells engineered tosecrete human granulocyte-macrophage colony-stimulating factor generatespotent antitumor immunity in patients with metastatic melanoma. PNAS95(22):13141-13146 (1998)). Finally, Bendandi et al. demonstratedmolecular remission of residual lymphoma in 8/11 patients aftertreatment with an idiotype protein vaccine in combination with either100 or 500 ug/M2 of GM-CSF (Bendandi et al. Complete molecularremissions induced by patient-specific vaccination plusgranulocyte-monocyte colony-stimulating factor against lymphoma. NatureMedicine 5(10):1171-7 (1999)). Importantly, in this trial the vaccinewas given once per month for four cycles with a booster given 2 monthsafter the final cycle. GM-CSF was given at the time of vaccination anddaily, for three additional doses. These data suggest that in vivoadministration of GM-CSF may supplant the need for in vitro culture ofDC precursors.

Additional problems with whole tumor based approaches include thepotential for tumor contamination, small cell number, and limitedability to monitor the immune response. An alternative approach is theuse of tumor associated synthetic antigens for immunologic priming.Peptide-based strategies for DC priming enable prior characterization ofthe immunologic stimulant, facilitating subsequent analysis of theanti-tumor immune response. Because specific peptides are ubiquitous intumors of the same histologic type, identical peptide vaccines may beemployed in allogeneic hosts bearing the same tumor histology.Additionally, the use of single peptides for immunization limits thepotential induction of undesired autoimmunity (Nestle et al. Vaccinationof melanoma patients with peptide- or tumor lysate-pulsed dendriticcells. Nature Medicine 4(3):328-32 (1998); Tsai et al. In vitroimmunization and expansion of antigen-specific cytotoxic T lymphocytesfor adoptive immunotherapy using peptide-pulsed dendritic cells.Critical Reviews in Immunology 18(1-2):65-75 (1998); Tsai et al.Identification of subdominant CTL epitopes of the GP100melanoma-associated tumor antigen by primary in vitro immunization withpeptide-pulsed dendritic cells. Journal of Immunology 158(4):1796-1802(1997)). Finally, recent developments in the use of soluble MHC Class Ipeptide tetramers/dimers and Elispot technology have enabled rapidcharacterization of epitope specific CTL response (Altman et al.Phenotypic analysis of antigen-specific T lymphocytes. Science274(5284):94-96 (1996); V. Cerundolo. Use of major histocompatibilitycomplex class I tetramers to monitor tumor-specific cytotoxic Tlymphocyte response in melanoma patients. Cancer Chemotherapy &Pharmacology 46(Suppl):S83-5 (2000)). The primary limitations topeptide-based vaccine strategies are haplotype restriction; potentialfor degradation; the lack of identifiable putative tumor antigensrecognized to induce a CTL response; the potential failure of theefferent arm of the immune response if smaller numbers of peptides areemployed; and uncertainty regarding which peptides, used alone or incombination, are the most immunogenic (Nair et al. Regression of tumorsin mice vaccinated with professional antigen-presenting cells pulsedwith tumor extracts. International Journal of Cancer 70(6):706-15(1997); Amoscato et al. Rapid extracellular degradation of syntheticclass I peptides by human dendritic cells. Journal of Immunology161(8):4023-32 (1998)).

The optimal antigenic target is derived from a protein which isessential for cell survival, is expressed on all tumor cells, is tumorspecific, is a surface protein, and is not expressed in the thymus norduring fetal development. No protein identified to date satisfies all ofthese criteria with regard to SCCHN tumors. However, several proteinshave been identified with peptide epitopes capable of stimulatingantigen specific CTL against SCCHN including SART1, SART 3, CASP8, andSCCA 1 (Hamada et al. Molecular cloning of human squamous cell carcinomaantigen 1 gene and characterization of its promoter, Biochimica etBiophysica Acta 1518(1-2):124-31 (2001); Nakao et al. Identification ofa gene coding for a new squamous cell carcinoma antigen recognized bythe CTL. Journal of Immunology 164(5):2565-74 (2000); Shichijo et al. Agene encoding antigenic peptides of human squamous cell carcinomarecognized by cytotoxic T lymphocytes. J. of Exp. Med. 187(3):277-88(1998); Yang et al. Identification of a gene coding for a proteinpossessing shared tumor epitopes capable of inducing HLA-A24-restrictedcytotoxic T lymphocytes in cancer patients. Cancer Research59(16):4056-63 (1999)). The major limitations to clinical application ofthese peptide epitopes are both their limited prevalence in SCCHN andputative epitopes restricted by HLA types with low population specificfrequencies. For example, while SART-1 is expressed in the majority ofSCCHN, the defined peptide epitopes are HLA26 restricted, limitingpotential therapeutic application (Shichijo et al. A gene encodingantigenic peptides of human squamous cell carcinoma recognized bycytotoxic T lymphocytes. J. of Exp. Med. 187(3):277-88 (1998)). In orderto increase therapeutic application, proteins should optimally beexpressed in the majority of SCCHN, with defined epitopes representativeof common HLA haplotypes.

One attractive candidate antigenic target for use in treating SCCHN isthe MAGE-A3 differentiation antigen (SEQ ID NO: 27) initially identifiedin the human MZ2E melanoma (van der Bruggen et al. A gene encoding anantigen recognized by cytolytic T lymphocytes on a human melanoma.Science 254(5038):1643-7 (1991)). This protein is a member of the cancertestis family and is expressed on tumors of diverse histologic types.MAGE-A3 has high potential utility for the immunotherapy of SCCHN basedon its tumor specificity, high percent expression, and the existence ofpreviously defined epitopes. A recent report demonstrated the presenceof MAGE-A3 in 44.4% of freshly isolated SCCHN by PCR and in 27% ofspecimens by immunohistochemistry (Kienstra et al. Identification ofNY-ESO-1, MAGE-1, and MAGE-3 in head and neck squamous cell carcinoma.Head and Neck 25(6):457-463 (2003)). Additionally, HLA-A2 epitopes havebeen identified and clinically evaluated for the treatment ofgastrointestinal malignancies and melanomas. Specifically, Sadanaga etal. utilized DC pulsed with the FLWGPRALV peptide (SEQ ID NO:1),restricted to the HLA-A2 epitope (van der Bruggen et al. A peptideencoded by human gene MAGE-3 and presented by HLA-A2 induces cytolytic Tlymphocytes that recognize tumor cells expressing MAGE-3. EuropeanJournal of Immunology 24(12):3038-43 (1994)), and demonstrated theinduction of CTL in 2/5 patients, with 2/6 patients enjoying a mixedclinical response (Sadanaga et al. Dendritic cell vaccination with MAGEpeptide is a novel therapeutic approach for gastrointestinal carcinomas.Clinical Cancer Research 7(8):2277-2284 (2001)). In a similar study inpatients with metastatic melanoma, patients were vaccinated with PBMCpulsed with either MAGE-A3 or MelanA peptides in combination with IL-12.In eight patients who demonstrated an increased immune response, therewas one complete response, one partial response, one minor response, andtwo mixed responses. Interestingly, in the mixed responders, tumorspecimens that did not respond to treatment did not express the antigenused for vaccination (Gajewski et al. Immunization of HLA-A2+ melanomapatients with MAGE-3 or MelanA peptide-pulsed autologous peripheralblood mononuclear cells plus recombinant human interleukin 12. ClinicalCancer Research 7(3 Suppl):895s-901s (2001)).

Recently, a new peptide epitope of MAGE-A3, KVAELVHFL (SEQ ID NO:2), hasbeen defined (Kawashima et al. The multi-epitope approach forimmunotherapy for cancer: identification of several CTL epitopes fromvarious tumor-associated antigens expressed on solid epithelial tumors.Human Immunology 59(1):1-14 (1998)). Dendritic cells pulsed with thispeptide stimulate naïve CTL to lyse MAGE-A3 positive tumors with anHLA-A2.1 phenotype. Although this peptide has not been clinicallyevaluated, studies suggest that it is capable of stimulating a higherpercentage of tumor reactive CTL than FLWGPRALV.

In addition to class I epitopes, immunogenic HLA-DR restricted class IIepitopes have also been defined for MAGE-A3. Specifically, Chaux et al.have identified an HLA-DR13 MAGE-A3₁₁₄₋₁₂₇ epitope AELVHFLLLKYRAR (SEQID NO:3), recognized by epitope specific HTL clones (Chaux et al.Identification of MAGE-3 epitopes presented by HLA-DR molecules toCD4(+) T lymphocytes. Journal of Experimental Medicine 189(5):767-7781999)). Similarly, Manici et al. used a bioinformatics based approach tocharacterize 3 MAGE-A3 epitopes for HLA-DR11, MAGE-A3₂₈₁₋₂₉₅,MAGE-A3₁₄₁₋₁₅₅, and MAGE-A3₁₄₆₋₁₆₀. T cells stimulated withMAGE-A3₂₈₁₋₂₉₅, TSYVKVLHHMVKISG (SEQ ID NO:4), were capable of lysingHLA-DR11/MAGE-A3 positive melanomas (Manici et al. Melanoma cellspresent a MAGE-3 epitope to CD4(+) cytotoxic T cells in association withhistocompatibility leukocyte antigen DR11. Journal of ExperimentalMedicine 189(5):871-876 (1999)). Additional recent evaluations by thepresent inventors demonstrated MAGE-A3₁₄₆₋₁₆₀ (VIFSKASSSLQL; SEQ IDNO:5) can stimulate T-helper cells restricted by both HLA-DR4 andHLA-DR7. MAGE-A3₁₄₆₋₁₆₀ is naturally processed, as peptide stimulated Tcells react with DC primed with whole tumor preparations (Kobayashi etal. Tumor-reactive T helper lymphocytes recognize a promiscuous MAGE-A3epitope presented by various major histocompatibility complex class IIalleles. Cancer Research 61(12):4773-8 (2001)). Based on these twostudies, it is clear that MAGE-A3₁₄₆₋₁₆₀ is naturally processed peptideepitope and that it is promiscuous for multiple HLA-DR epitopes, makingit an ideal candidate for therapeutic application.

A second attractive candidate for peptide-based immunotherapy is thehuman papilloma virus (HPV) 16 E7 nuclear protein (SEQ ID NO:28). HPV 16E7 protein is a tumor rejection antigen, which is postulated to play anintegral role in the development of carcinoma of the uterine cervix.Recent studies by the present inventors, as well as others, haveidentified HPV 16 as an independent risk factor for oropharyngeal SCC(Strome et al. Squamous Cell Carcinoma of the Tonsils: A MolecularAnalysis of HPV Associations, Clinical Cancer Research 18:1093-1100(2002); Gillison et al. Evidence for a casual association between humanpapillomavirus and a subset of head and neck cancers. Journal of theNational Cancer Institute 92:709-720 (2000); Gillison et al. Humanpapillomavirus in head and neck squamous cell carcinoma: are some headand neck cancers a sexually transmitted disease? Current Opinion inOncology 11:191-199 (1999)). While a cause effect relationship remainsto be established, several in vitro studies suggest that continuedexpression of the nuclear E6 and E7 proteins is likely required formalignant transformation of infected cells (Crook et al. Continuedexpression of HPV-16 E7 protein is required for maintenance of thetransformed phenotype of cells co-transformed by HPV-16 plus EJ-ras.EMBO Journal 8(2):513-9 (1989); Munger et al. The E6 and E7 genes of thehuman papillomavirus type 16 together are necessary and sufficient fortransformation of primary human keratinocytes. Journal of Virology63(10):4417-21 (1989)). Because of its association with malignancies ofdiverse histologic types, development of HPV-based peptide immunotherapyplatforms can potentially impact the treatment of multiple diseaseentities.

Several groups have now identified HLA-A2 and HLA-DR restrictedantigenic epitopes for the HPV 16 E7 nuclear protein (Feltkamp et al.Vaccination with cytotoxic T lymphocyte epitope-containing peptideprotects against a tumor induced by human papillomavirus type16-transformed cells. European Journal of Immunology 23(9):2242-2249(1993); Feltkamp et al. Cytotoxic T lymphocytes raised against asubdominant epitope offered as a synthetic peptide eradicate humanpapillomavirus type 16-induced tumors. European Journal of Immunology25(9):2638-42 (1995); Chen et al. Human papillomavirus type 16nucleoprotein E7 is a tumor rejection antigen. PNAS USA 88(1):110-4(1991); Nijman et al. Characterization of cytotoxic T lymphocyteepitopes of a self-protein, p53, and a non-self-protein, influenzamatrix: relationship between major histocompatibility complex peptidebinding affinity and immune responsiveness to peptides. Journal ofImmunotherapy 14(2):121-6 (1993)). Two of these epitopes, E7 12-20(MLDLQPETT; SEQ ID NO:6) and E7 86-93 (TLGIVCPI; SEQ ID NO:7), have beenevaluated in phase I trials for cervical carcinoma. Treatment of 18women who were HLA-A2 positive with high-grade cervical intraepithelialneoplasia with this regimen resulted in 3 complete responses and 6partial responses (Nijman et al. Characterization of cytotoxic Tlymphocyte epitopes of a self-protein, p53, and a non-self-protein,influenza matrix: relationship between major histocompatibility complexpeptide binding affinity and immune responsiveness to peptides. Journalof Immunotherapy 14(2):121-6 (1993); Muderspach et al. A phase I trialof a human papillomavirus (HPV) peptide vaccine for women withhigh-grade cervical and vulvar intraepithelial neoplasia who are HPV 16positive. Clin Cancer Res 6(9):3406-16 (2000)). Additionally, Van derBurg et al. have recently defined an HPV 16 E7 helper epitope(PAGQAEPDRAHYNIVTFCCKCD; SEQ ID NO:8) which effectively stimulates CD4responses in patients with HPV 16 positive cervical lesions (van derBurg et al. Natural T-helper immunity against human papillomavirus type16 (HPV16) E7-derived peptide epitopes in patients with HPV16-positivecervical lesions: identification of 3 human leukocyte antigen classII-restricted epitopes. International Journal of Cancer 91(5):612-8(2001)).

Potential pitfalls in the development of peptide-based immunotherapy forSCCHN including: 1) peptide-induced tolerance, 2) synthetic peptidedegradation, 3) limited antigenic repertoire, and 4) inadequate toolsfor evaluating treatment response. It is now clear that depending on thedose and timing of drug delivery, the same peptide can induce eitherantigen specific CTL or T cell deletion (Zinkemagel et al. Antigenlocalisation regulates immune responses in a dose- and time-dependentfashion: a geographical view of immune reactivity. Immunological Reviews156:199-209 (1997); Mullbacher et al. In vivo administration of majorhistocompatibility complex class I-specific peptides from influenzavirus induces specific cytotoxic T cell hyporesponsiveness. EuropeanJournal of Immunology 23(10):2526-31 (1993); Gallimore et al.Hierarchies of antigen-specific cytotoxic T-cell responses.Immunological Reviews 164:29-36 (1998); Aichele et al. T cell primingversus T cell tolerance induced by synthetic peptides. Journal ofExperimental Medicine 182(1):261-6, 1995)). The primary factors indetermining the type of peptide-induced T cell response appear to belargely pharmacokinetic in nature (Weijzen et al. Pharmacokineticdifferences between a T cell-tolerizing and a T cell-activating peptide.J Immunol 166(12):7151-7 (2001)). Specifically, peptides which achievehigh initial tissue concentrations followed by rapid elimination tend toinduce T cell deletion. Importantly, recent studies by the presentinventors have clearly demonstrated that systemic peptideadministration, recognized to induce T cell deletion, is preceded by Tcell proliferation. In contrast, peptides that achieve gradual tissueuptake and maintain their presence preferentially induce a CTL response.In order to limit the potential for rapid systemic antigen exposure inthis study, an approved human adjuvant (Montanide ISA 51), which issimilar to incomplete Freunds adjuvant (IFA), may be used to regulatethe temporal aspects of peptide release (Aichele et al. T cell primingversus T cell tolerance induced by synthetic peptides. Journal ofExperimental Medicine 182(1):261-6, 1995)).

The second potential problem with synthetic peptide-based immunotherapyis the potential for proteolysis. A recent report by Amoscato et al. hasclearly demonstrated that DC induces peptide degradation through bothendo- and ectoproteolysis. Ectocellular DC mediated proteolysis isprimarily mediated through CD13, a molecule which appears to play aphysiologic role in DC migration and extracellular antigen processing(Amoscato et al. Rapid extracellular degradation of synthetic class Ipeptides by human dendritic cells. Journal of Immunology 161(8):4023-32(1998)). Several strategies have been designed to overcome thephysiologic degradation of synthetic peptides including N and C terminalmodifications and dose increases (Amoscato et al. Rapid extracellulardegradation of synthetic class I peptides by human dendritic cells.Journal of Immunology 161(8):4023-32 (1998)). While modification of theamino terminus has been demonstrated to reduce degradation and enhancepresentation of a class II epitope, there are concerns that peptidemodification can alter HLA binding (Dong et al. Modification of theamino terminus of a class II epitope confers resistance to degradationby CD13 on dendritic cells and enhances presentation to T cells. Journalof Immunology 164(1):129-35 (2000)). Additionally, as previouslymentioned, while increased doses may enhance peptide availability forpresentation, in specific settings, alteration of peptidepharmacokinetics can induce T cell deletion.

An alternative means to overcome the problem of proteolysis is throughthe construction of long “Trojan antigens.” The present inventors haverecently demonstrated that large synthetic peptides, up to 50 aminoacids in length, which contain multiple epitopes linked to atranslocating region of HIV TAT (RKKRRQRRR; SEQ ID NO:9) can beinternalized and processed. Additionally, these peptides appear to behighly resistant to proteolysis and do not require proteosomalprocessing and transport by TAP, since they penetrate directly to the ERand Golgi where they form peptide/MHC complexes (Lu et al.TAP-independent presentation of CTL epitopes by Trojan antigens. Journalof Immunology 166(12):7063-71 (2001)). The present inventors have alsoestablished that multiple T cell epitopes can be joined together usingfurin-cleavable linkers (RVKR; SEQ ID NO:10), which allow the release ofthe individual epitopes in the Golgi, where the furin endopeptidaseresides.

The third potential limiting factor for peptide-based immunotherapy isrelated to a defined antigenic repertoire that is HLA restricted. Thisfactor, inherent to all peptide-based approaches, restricts patientaccess. Additionally, because individual peptides only have thepotential to induce epitope specific CTL, the vast majority of potentialtumor antigens are not targeted. In this setting, tumor down regulationof individual antigens or HLA epitopes promotes immune evasion. Recentevidence, however, suggests that this problem of epitope restriction maynot be as physiologically important as was previously postulated.Specifically, it has now been clearly demonstrated that a T cellresponse induced against one epitope can stimulate CTL response to othertarget epitopes through a mechanism termed epitope-spreading (Vanderlugtet al. Epitope spreading in immune-mediated diseases: implications forimmunotherapy. Nature Reviews. Immunology 2(2):85-95 (2002)). Using anexperimental autoimmune encephalitis model, Vanderlugt et al. havedemonstrated that disease progression is associated with the developmentof epitope specific helper T cells which are distinct from thoseinitiating the disease. Transfer of secondary CD4 cells to naïve miceinduces the disease phenotype and the disease is abrogated by blockingthe secondary T cell response even though the primary T cell responseremains intact (Prehn et al. Immunity to methylcolanthrene-inducedsarcomas. Journal of the National Cancer Institute 6:769-778 (1957);McRae et al. Functional evidence for epitope spreading in the relapsingpathology of experimental autoimmune encephalomyelitis. Journal ofExperimental Medicine 182(1):75-85 (1995)). These data suggest thatpeptide-based approaches to cancer immunotherapy may indirectlystimulate multiple tumor reactive CTL against minor antigens in thepresence of residual tumor.

The fourth and final limitation to peptide-based immunotherapy, is thelimited number of diagnostic tools available to evaluate clinicalresponse. Positron emission tomography (PET) in combination withsystemic administration of a glucose analogue, FDG, is a relatively newimaging modality that measures metabolic activity of individual tissues.Tissues with large energy requirements, e.g. tumors, incorporate higherlevels of FDG than surrounding normal tissue, allowing whole body tumorscreening. Because other pathogenic processes, such as infection, canhave high metabolic requirements, PET is not tumor specific. However, inpatients with known tumors, PET is highly accurate for both staging headand neck cancer and identifying recurrent disease (Rege et al. Use ofpositron emission tomography with fluorodeoxyglucose in patients withextracranial head and neck cancers, Cancer 73(12):3047-3058 (1994);McGuirt et al. A comparative diagnostic study of head and neck nodalmetastases using positron emission tomography. Laryngoscope 105((4 Pt1)): 373-375 (1995); Laubenbacher et al. Comparison offluorine-18-fluorodeoxyglucose PET, MRI and endoscopy for staging headand neck squamous-cell carcinomas. Journal of Nuclear Medicine36(10):1747-1757 (1995); Lapela et al. Head and neck cancer: detectionof recurrence with PET and 2-[F-18]fluoro-2-deoxy-D-glucose. Radiology197:205-211 (1995); Bailet et al. Positron emission tomography: a new,precise imaging modality for detection of primary head and neck tumorsand assessment of cervical adenopathy. Laryngoscope 102:281-288 (1992)).In fact, in one recent study, PET had a sensitivity of 88% compared to25% for MRI/CT for identifying recurrent head and neck malignancy (Anzaiet al. Recurrence of head and neck cancer after surgery or irradiation:prospective comparison of 2-deoxy-2-[F-18]fluoro-D-glucose PET and MRimaging diagnoses. Radiology 200(1):135-141 (1996)). In patients beingevaluated for response to chemotherapy, PET can accurately identifyresidual disease—even in some cases where initial biopsies are negative(Lowe et al. Prediction of Chemotherapy Response in Patients withAdvanced Head and Neck Cancer Using [18F] Fluoro-deoxyglucose PositronEmission Tomography (FDG-PET). Head and Neck 19:666-674 (1997)). Recentinnovations in PET technology that combine PET and CT machines into one,largely overcome the lack of anatomic detail traditionally hampering PETscan interpretation.

In view of the need for additional therapeutic options for use in thetreatment of SCCHN, especially in patients with unresectable disease,the present invention provides novel Trojan antigen-based compositionsand method for their use in the treatment of SCCHN. More specifically,MAGE-A3 and HPV 16-based Trojan antigen compositions, each composed of1-2 HLA-A2.1 restricted CTL epitopes, HLA-DR helper epitopes joinedtogether with furin-cleavable linkers and HIV TAT translocating region.

SUMMARY OF THE INVENTION

The present invention relates to Trojan antigens, and immunogeniccompositions comprising the Trojan antigens. The present invention alsorelates to methods of generating an immune response in a subject usingthe Trojan antigens or immunogenic compositions. The present inventionfurther relates to methods of treating squamous cell carcinoma of thehead and neck (SCCHN) using the Trojan antigens or immunogeniccompositions of the present invention.

More specifically, the present invention related to Trojan antigens,which include an isolated polypeptide comprising amino acids 1-35 of SEQID NO:15, an isolated polypeptide comprising amino acids 1-47 of SEQ IDNO:17, an isolated polypeptide comprising amino acids 1-21 of SEQ IDNO:19, and an isolated polypeptide comprising amino acids 1-43 of SEQ IDNO:22.

In the Trojan antigen of SEQ ID NO:19, X may be cysteine or aminobutyricacid. In the Trojan antigen of SEQ ID NO:22, each X may independently becysteine or aminobutyric acid.

The immunogenic compositions of the present invention includeimmunogenic compositions comprising one or more of the following Trojanantigens: an isolated polypeptide comprising amino acids 1-35 of SEQ IDNO:15, an isolated polypeptide comprising amino acids 1-47 of SEQ IDNO:17, an isolated polypeptide comprising amino acids 1-21 of SEQ IDNO:19, wherein X may be cysteine or aminobutyric acid, and an isolatedpolypeptide comprising amino acids 1-43 of SEQ ID NO:22, wherein X₆,X₃₀, X₃₁ and X₃₃ are each independently cysteine or aminobutyric acid.The immunogenic compositions of the present invention further comprise apharmaceutically acceptable carrier, diluent or adjuvant.

The methods of generating an immune response in a subject of the presentinvention comprising administering one or more of the following Trojanantigens to a subject in an amount sufficient to induce an immuneresponse in said subject: a polypeptide comprising amino acids 1-35 ofSEQ ID NO:15, a polypeptide comprising amino acids 1-47 of SEQ ID NO:17,a polypeptide comprising amino acids 1-21 of SEQ ID NO:19, wherein X maybe cysteine or aminobutyric acid, and a polypeptide comprising aminoacids 1-43 of SEQ ID NO:22, wherein X₆, X₃₀, X₃₁ and X₃₃ are eachindependently cysteine or aminobutyric acid. The one or more Trojanantigens may be co-administered with a pharmaceutically acceptablecarrier, diluent or adjuvant.

In a preferred embodiment, the present invention includes a method ofgenerating an immune response in a subject comprising administering thefollowing Trojan antigens to a subject in an amount sufficient to inducean immune response in said subject: (a) a polypeptide comprising aminoacids 1-47 of SEQ ID NO:17 and (b) a polypeptide comprising amino acids1-43 of SEQ ID NO:22, wherein X₆, X₃₀, X₃₁ and X₃₃ are eachindependently cysteine or aminobutyric acid. The Trojan antigens may beco-administered with a pharmaceutically acceptable carrier, diluent oradjuvant.

In preferred embodiments, the one or more Trojan antigens areadministered in a combined amount of between about 100 ug and about 1.5mg, more preferably in an amount of about 1 mg.

In other preferred embodiments, the one or more Trojan antigens areco-administered with montanide, in an amount of between about 0.5 and1.5 mL, and GM-CSF, in an amount of between about 50 and 150 ug/m².

The methods treating squamous cell carcinoma of the head and neck(SCCHN) of the present invention comprising administering to a subjectin need of such treatment a therapeutically-effective amount of one ofthe following Trojan antigens: a polypeptide comprising amino acids 1-35of SEQ ID NO:15, a polypeptide comprising amino acids 1-47 of SEQ IDNO:17, a polypeptide comprising amino acids 1-21 of SEQ ID NO:19,wherein X may be cysteine or aminobutyric acid, and a polypeptidecomprising amino acids 1-43 of SEQ ID NO:22, wherein X₆, X₃₀, X₃₁ andX₃₃ are each independently cysteine or aminobutyric acid. The one ormore Trojan antigens may be co-administered with a pharmaceuticallyacceptable carrier, diluent or adjuvant.

In a preferred embodiment, the method of treating SCCHN comprisesadministering to a subject in need of such treatment atherapeutically-effective amount of the following Trojan antigens: (a) apolypeptide comprising amino acids 1-47 of SEQ ID NO:17 and (b) apolypeptide comprising amino acids 1-43 of SEQ ID NO:22, wherein X₆,X₃₀, X₃₁ and X₃₃ are each independently cysteine or aminobutyric acid.The Trojan antigens may be co-administered with a pharmaceuticallyacceptable carrier, diluent or adjuvant.

In preferred embodiments, the one or more Trojan antigens areadministered in a combined amount of between about 100 ug and about 1.5mg, more preferably in an amount of about 1 mg.

In other preferred embodiments, the one or more Trojan antigens areco-administered with montanide, in an amount of between about 0.5 and1.5 mL, and GM-CSF, in an amount of between about 50 and 150 ug/m².

The present invention also comprises the following polynucleotidemolecules: a polynucleotide molecule encoding amino acids 1-35 of SEQ IDNO:15, a polynucleotide molecule encoding amino acids 1-47 of SEQ IDNO:17, a polynucleotide molecule encoding amino acids 1-21 of SEQ IDNO:19, and a polynucleotide molecule amino acids 1-43 of SEQ ID NO:22.

The present invention further comprises expression vectors comprisingone of the following polynucleotide molecules: a polynucleotide moleculeencoding amino acids 1-35 of SEQ ID NO:15, a polynucleotide moleculeencoding amino acids 1-47 of SEQ ID NO:17, a polynucleotide moleculeencoding amino acids 1-21 of SEQ ID NO:19, and a polynucleotide moleculeamino acids 1-43 of SEQ ID NO:22.

The present invention additionally includes a host cell comprising anexpression vector, wherein the expression vector comprises one of thefollowing polynucleotide molecules: a polynucleotide molecule encodingamino acids 1-35 of SEQ ID NO:15, a polynucleotide molecule encodingamino acids 1-47 of SEQ ID NO:17, a polynucleotide molecule encodingamino acids 1-21 of SEQ ID NO:19, and a polynucleotide molecule aminoacids 1-43 of SEQ ID NO:22.

The present invention further includes methods of preparing apolypeptide, comprising culturing a host cell comprising an expressionvector, which in turn comprises a polynucleotide molecule, underconditions promoting expression of the polypeptide encoded by thepolynucleotide molecule and recovering the polypeptide from the cellculture. In preferred embodiments, the polynucleotides include: apolynucleotide molecule encoding amino acids 1-35 of SEQ ID NO:15, apolynucleotide molecule encoding amino acids 1-47 of SEQ ID NO:17, apolynucleotide molecule encoding amino acids 1-21 of SEQ ID NO:19, and apolynucleotide molecule amino acids 1-43 of SEQ ID NO:22.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graphical representation of the ability of a MAGE3-specificcytotoxic T lymphocyte (CTL) clone to recognize different tumor types.An MAGE3[9₁₁₂]-specific CTL clone was tested for cytotoxicity using thefollowing targets: ∘, .221A2.1 pulsed with MAGE3[9₁₁₂], ∘, .221A2.1without peptide; Δ, 624mel (melanoma, A2⁺, MAGE3⁺); □, KATO-III (gastricCa, A2⁺, MAGE3⁺); ⋄, SW403 (colon Ca, A2⁺, MAGE3⁺); □, WiDr (colon Ca,A2⁻, MAGE3⁺); Δ, 888mel (melanoma, A2⁻, MAGE3⁻).

FIG. 2 is the results of experiments that demonstratedHLA-DR4-restricted HTL clone 8G9 recognizes naturally processed MAGE A3antigen. A: Proliferative T-cell response induced by MAGE-A3₁₄₆₋₁₆₀(+Peptide), recombinant MAGE-A3 protein (rMAGE-A3) or recombinant gp100(rgp 100). B: Tissue culture supernatants from experiment described inpanel A, were collected after 48 hr and the concentration of GM-CSF wasmeasured by ELISA. C: T-cell clone 8G9 recognizes UV-irradiated melanomacells that express MAGE-A3 (HT-144 (+), SKmel-28 (+), 697mel (+)) viaantigen cross-presentation by autologous DC. DC incubated with MAGE-A3negative melanoma cell line (888mel (−)) did not stimulate the T-cellclone. Values shown are the means of triplicate determinations; bars,SD.

FIG. 3 is the result of an experiment where dendritic cells where pulsedwith a Trojan antigen. C57/BL6 DC were pulsed with either SIINFEKL (SEQID NO:11) or TrojAg (RKKRRQRRRRAAASIINFEKL; SEQ ID NO:12) for 2 hours,washed three times, and then kept in 37° C. After 48 hours, the abilityof peptide loaded DC to induce IFN-gamma release from OT-1 T cells wasdetermined by ELISA. The concentration of IFN-gamma was determined atvarious dilutions of the supernatant that was collected after 24 hoursof incubation of the DC with the OT-1 T cells.

FIG. 4 shows the results of an Elispot analysis of IFN γ production byHPV 16 Trojan antigen stimulated T cells.

FIG. 5 shows the results of an immunohistochemical analysis of HLA-A2expression in fresh SCCHN. Panel A: HLA-A2 negative patient with nostaining of the tumor or surrounding parenchyma. Panel B: HLA-A2positive patient with positive staining of both the tumor andsurrounding parenchyma, arrows indicate tumor location. Panel C: HLA-A2positive patient with positive staining of the parenchyma, but loss ofHLA-A2 reactivity in the tumor. Specimens were stained with H&E(hematoxylin and eosin), mouse Ig control or the SB03-111 antibody.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to Trojan antigens, and immunogeniccompositions comprising the Trojan antigens. The present invention alsorelates to methods of generating an immune response in a subject usingthe Trojan antigens or immunogenic compositions. The present inventionfurther relates to methods of treating squamous cell carcinoma of thehead and neck (SCCHN) using the Trojan antigens or immunogeniccompositions of the present invention.

Trojan Antigens

One embodiment of the present invention pertains to Trojan antigens.Trojan antigens are polypeptides comprising one or more antigenicepitopes joined together by cleavable linkers, that may be used aspeptide vaccines for administration to a subject. Therapeutic peptidevaccines may be used to induce a subject's innate anti-tumor response byusing antigenic epitopes derived from polypeptides expressed by thetumor cells from said subject. Trojan antigens are processed by antigenpresenting cells (APC) which then display the antigenic epitopes of theTrojan antigens on their surface. Cytotoxic T lymphocytes (CTL) areactivated by the APC displaying the antigenic epitopes in the context ofMHC class I molecules, which then recognize and destroy tumor cellsdisplaying the antigenic epitope in the context of a larger polypeptide.

In addition to the activation of CTL, a CD4+ helper T cell response isalso activated by the APC displaying the antigenic epitopes in thecontext of MHC class II molecules.

In addition to the antigenic epitopes derived from tumor-expressedproteins, the Trojan antigens of the present invention may also includea transporter peptide. Transporter peptides are regions of polypeptidesknown to be translocated into cells without first requiring proteosomalprocessing and transport by TAP (transporter associated with antigenprocessing). Polypeptides comprising transporter peptides areinternalized directly to the endoplasmic reticulum (ER) and Golgi.Inclusion of a transporter peptide in the Trojan antigens allows theTrojan antigens to be directly taken up by APC where the antigenicepitopes can form peptide/MHC complexes. Such Trojan antigens are moreresistant to degradation than smaller constructs and allow simultaneousstimulation of multiple T cell populations, reducing the chance of tumorescape through the selection of antigen loss variants.

The antigenic epitopes are portions of a polypeptide, or the entirepolypeptide in the case of small proteins, expressed by a tumor cell.Preferably, the polypeptide is essential for cell survival, is expressedby all cells of the tumor, is tumor specific, is a surface protein, andis a protein not expressed in the thymus nor during fetal development.After uptake and processing of Trojan antigens by antigen presentingcells, the antigenic epitopes are displayed on the cell surface in thecontext of MHC class I and class II molecules, which in turn induce CD8+and CD4+ T cells, respectively. The Trojan antigens of the presentinvention may comprise antigenic epitopes that include CD8+ T cellsalone, CD4+ T cells alone, or both CD8+ and CD4+ T cells.

While the composition of the antigenic epitopes is governed by thefactors noted above, peptides homologous to antigenic epitopes may alsobe used in Trojan antigens to increased the spectrum of immune responsegenerated by the Trojan antigens. Peptide homologues having at leastabout 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequenceidentity to a selected antigenic epitope are included in the presentinvention. Each reference to an antigenic epitope herein is also meantto be a reference to a peptide homologue that may be used in place ofthe antigenic epitope. A single Trojan antigen may comprise both aspecific antigenic epitope and a peptide homologue of the specificantigenic epitope.

The antigenic epitopes of the present invention may be comprised ofnaturally occurring amino acids or amino acid analogues. Such analoguesinclude those amino acids that produce cleavage-resistant peptides.

The size of the antigenic epitopes used in the Trojan antigens is notlimited, though preferably the antigenic epitopes are of a size thatreadily forms a complex with MHC class I and class II molecules inantigen presenting cells. Preferably, the antigenic epitopes for class Imolecules are peptides of between about 8 amino acids and about 10 aminoacids in length. More preferably, the antigenic epitopes for class Imolecules are peptides of about 9 amino acids in length. Preferably, theantigenic epitopes for class II molecules are peptides of between about10 amino acids and about 20 amino acids in length. More preferably, theantigenic epitopes for class II molecules are peptides of about 15 aminoacids in length.

The use of cleavable linkers to join antigenic epitopes (when two ormore antigenic epitopes are used in a Trojan antigen) or one or moreantigenic epitopes and the transporter peptide, allows the release ofthe individual components of the Trojan antigen upon internalization ofthe antigen into a cell.

Preferably, the cleavable linkers are furin-sensitive linkers whichallow the components of the Trojan antigen to be separated from eachother in the Golgi, where the furin endopeptidase resides. Furin is avery specific protease that recognizes a motif consisting of RX(R/K)R,where R and K are the positive-charged amino acids lysine and arginine,respectively, and X is any amino acid residue. A preferred furin linkerused herein is RVKR (SEQ ID NO:10). Through the action of furin in theGolgi, the Trojan antigen is first cleaved separating the antigenicepitopes from the transporter peptide. The antigenic epitopes are thentrimmed via amino- and carboxy-peptidases that are present in the ER andGolgi, until the appropriately-sized peptide is formed and binds to MHCmolecules in these compartments, protecting it from further degradation.The skilled artisan will understand that any furin-cleavable linker maybe used, where the first, third and fourth residues are positivelycharged amino acids such as lysine and arginine. Other suitable linkerswill be readily apparent to the skilled artisan.

The portion of the Trojan antigen that allows direct internalization ofthe antigens into a cell is a transporter peptide. The transporterpeptide is any peptide that allows transport of the Trojan antigendirectly into a cell without first requiring proteolytic processing ofthe antigen. An example of a transporter peptide is the penetrin peptideof HIV TAT (Frankel et al. Cellular uptake of the tat protein from humanimmunodeficiency virus. Cell 55:1189-1193 (1988)). Several other cellpenetrating sequences have been described from proteins, including theVP22 protein of herpes simplex virus (Elliott et al. Intercellulartrafficking and protein delivery by a herpesvirus structural protein.Cell 88:223-233 (1997); Phelan et al. Intercellular delivery offunctional p53 by the herpesvirus protein VP22. Nat. Biotechnol.16:440-443 (1998)), the fibroblast growth factor (Lin et al. Inhibitionof nuclear translocation of transcription factor NF-kappa B by asynthetic peptide containing a cell membrane-permeable motif and nuclearlocalization sequence. J. Biol. Chem. 270:14255-14258 (1995); Rojas etal. Genetic engineering of proteins with cell membrane permeability.Nat. Biotechnol. 16:370-375 (1998)) and the Drosophila Antennapediahomeodomain protein (Schutze-Redelmeier et al. Introduction of exogenousantigens into the MHC class I processing and presentation pathway byDrosophila antennapedia homeodomain primes cytotoxic T cells in vivo. J.Immunol. 157:650 (1996)).

Preferably, the transporter protein is the penetrin peptide of HIV TAT:RKKRRQRRR (SEQ ID NO:9). The transporter peptide may be coupled toeither the amino- or the carboxy-terminal end of the antigenic epitopes.

In one embodiment of the invention, the Trojan antigen is based on oneor more antigenic epitopes from the MAGE-A3 differentiation antigenlinked to a transporter peptide. MAGE-A3 is expressed by cells of SCCHNtumors. When more than one antigenic epitope from MAGE-A3 is used, theepitopes are linked by a cleavable linker, preferably a furin-sensitivelinker. In this embodiment, a cleavable linker is also used to link theselected MAGE-A3 antigenic epitopes to the transporter peptide. In apreferred embodiment, the MAGE-A3₁₁₂₋₁₂₀ antigenic epitope KVAELVHFL(SEQ ID NO:2) is linked to the MAGE-A3₂₇₁₋₂₇₉ antigenic epitopeFLWGPRALV (SEQ ID NO:1) using the furin-sensitive linker RVKR (SEQ IDNO:10), to produce the linked peptide KVAELVHFL-RVKR-FLWGPRALV (SEQ IDNO:13). Upon action by furin, the linked peptide is cleaved intoKVAELVHFLRVKR (SEQ ID NO:14) and FLWGPRALV (SEQ ID NO:1) in the Golgi.KVAELVHFLRVKR (SEQ ID NO:14) is then trimmed by exopeptidases into theMHC-binding peptide KVAELVHFL (SEQ ID NO:2).

In this preferred embodiment, the linked peptide KVAELVHFLRVKRFLWGPRALV(SEQ ID NO:13) is joined to the penetrin transporter peptide from HIVTAT: RKKRRQRRR (SEQ ID NO:9). In this embodiment, the Trojan antigen isKVAELVHFLRVKRFLWGPRALVRVKRRKKRRQRRR (SEQ ID NO:15). Trojan antigenscomprising the MAGE-A3₁₁₂₋₁₂₀ antigenic epitope linked by afurin-sensitive linker to HIV TAT penetrin (SEQ ID NO:9), or theMAGE-A3₂₇₁₋₂₇₉ antigenic epitope linked by a furin-sensitive linker toHIV TAT penetrin (SEQ ID NO:9), are also include in this invention.

In a further preferred embodiment of the invention, the Trojan antigenis comprised of antigenic epitopes from MAGE-A3 that induce both CD8+and CD4+ T cells responses, i.e., the antigenic epitopes induce theformation of both class I and class II MHC complexes by APC. In thispreferred embodiment, the MAGE class I antigenic epitopes MAGE-A3₁₁₂₋₁₂₀(KVAELVHFL; SEQ ID NO:2) and MAGE-A3₂₇₁₋₂₇₉ (FLWGPRALV; SEQ ID NO:1) arelinked with the class II MAGE-A3₁₄₉₋₁₆₀ antigenic epitope (VIFSKASSSLQL(SEQ ID NO:5) using the furin-sensitive linker RVKR (SEQ ID NO:10), toproduce the linked peptide KVAELVHFLRVKRFLWGPRALVRVKRVIFSKASSSLQL (SEQID NO:16).

In this preferred embodiment, the linked peptide (SEQ ID NO:16) isjoined to the penetrin transporter peptide from HIV TAT: RKKRRQRRR (SEQID NO:9). In this embodiment, the Trojan antigen isKVAELVHFLRVKRFLWGPRALVRVKRVIFSKASSSLQL-RKKRRQRRR (SEQ ID NO:17).

Trojan antigens comprising the MAGE-A3₁₄₉₋₁₆₀ antigenic epitope linkedby a furin-sensitive linker to HIV TAT penetrin (SEQ ID NO:9) are alsoinclude in this invention, as are Trojan antigens comprising theMAGE-A3₁₁₂₋₁₂₀ (SEQ ID NO:2) and MAGE-A3₁₄₉₋₁₆₀ (SEQ ID NO:5) antigenicepitopes linked to each other and to HIV TAT penetrin (SEQ ID NO:9) by afurin-sensitive linkers. Trojan antigens comprising the MAGE-A3₂₇₁₋₂₇₉(SEQ ID NO:1) and MAGE-A3₁₄₉₋₁₆₀ (SEQ ID NO:5) antigenic epitopes linkedto each other and to HIV TAT penetrin (SEQ ID NO:9) by a furin-sensitivelinkers are also included in the present invention.

In a related embodiment of the invention, the Trojan antigen is based onone or more antigenic epitopes from the human papilloma virus (HPV) 16E7 nuclear protein. HPV 16 E7 protein is a tumor rejection antigen,which is postulated to play an integral role in the development ofcarcinoma of the uterine cervix. When more than one antigenic epitopefrom HPV 16 E7 is used, the epitopes are linked by a cleavable linker,preferably a furin-sensitive linker. A cleavable linker is further usedto link one or more HPV 16 E7 antigenic epitopes to a transporterpeptide. In a preferred embodiment, the HPV 16 E7₈₆₋₉₃ antigenic epitopeTLGIVXPI (SEQ ID NO:18), where X is cysteine or aminobutyric acid,preferably aminobutyric acid, is linked to the penetrin peptide sequencefrom HIV TAT: RKKRRQRRR (SEQ ID NO:9). In this embodiment, the Trojanantigen is TLGIVXPIRVKR-RKKRRQRRR (SEQ ID NO:19), where X is cysteine oraminobutyric acid, preferably aminobutyric acid.

In a preferred embodiment of an HPV 16 E7-based Trojan antigen, the HPV16 E7₈₆₋₉₃ antigenic epitope TLGIVXPI (SEQ ID NO:18) is linked via afurin-sensitive linker to the HPV 16 E7₄₁₋₆₂ antigenic epitopePAGQAEPDRAHYNIVTFXXKXD (SEQ ID NO:20), to form the linked peptideTLGIVXPIRVKRPAGQAEPDRAHYNIVTFXXKXD (SEQ ID NO:21), where each X isindependently cysteine or aminobutyric acid, preferably each X isaminobutyric acid. This linked peptide (SEQ ID NO:21) is joined to theHIV TAT transporter peptide RKKRRQRRR (SEQ ID NO:9) to create the Trojanantigen TLGIVXPIRVKRPAGQAEPDRAHYNIVTFXXKXDRKKRRQRRR (SEQ ID NO:22),where again each X is independently cysteine or aminobutyric acid,preferably each X is aminobutyric acid.

In addition to the HLA-A2 antigenic epitopes from the MAGE-A3 and HPV 16E7 polypeptides, antigenic epitopes from these two polypeptides thatassociate with alternative HLA alleles may be used in the presentinvention. Similarly, antigenic epitopes from other tumor expressedpolypeptides, such as telomerase, may be used in the present invention,both those that associate with HLA-A2, and those that associates withother HLA alleles.

The each of the antigenic epitopes, polypeptides, peptides, linkers andTrojan antigens of the present invention may be prepared based onmethods well known to those of skill in the art. For example, theseamino acid sequences can be produced by using recombinant DNA techniqueseasily identified and well known by those of skill in the art. Forexample, DNA molecules encoding the Trojan antigens are prepared usinggenerally available methods such as PCR mutagenesis, site-directedmutagenesis, and/or restriction digestion and ligation. The hybrid DNAis then inserted into expression vectors and introduced into suitablehost cells. Preferred expression vectors include plasmids and cosmids.An expression vector containing one or more polynucleotides encoding oneor more of the Trojan antigens of this invention can be used totransfect or transform a suitable host cell (prokaryotic or eukaryotic)to produce the protein or to produce an immune response, or for someother purpose.

A recombinant virus can also be used as the expression vector. Exemplaryviruses include the adenoviruses, adeno-associated viruses, herpesviruses, vaccinia, CMV, BLUESCRIPT (Stratagene, San Diego, Calif.),baculovirus, or an RNA virus such as a retrovirus or an alphavirus.Preferably, the retroviral vector is a derivative of a murine or avianretrovirus. The alphavirus vector is preferably derived from Sindbis orSemliki Forest Virus. All of these expression vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated.

The viral vector can be made target specific by inserting one or moresequences of interest into the viral vector, along with anotherpolynucleotide encoding a Trojan antigen. For example, retroviralvectors can be made target specific by inserting a polynucleotideencoding a sugar, a glycolipid, or a protein. Those of skill in the artwill know of, or can readily ascertain without undue experimentation,specific polynucleotide sequences which can be inserted into theretroviral genome to allow target specific delivery of the retroviralvector containing the polynucleotides of interest.

It will be appreciated that the same techniques that are utilized toincorporate the nucleotide sequences encoding a Trojan antigen, andoptionally other immunostimulatory polynucleotides, into viral geneexpression vectors can be used to incorporate the sequences into liveand attenuated live viruses for use as immunogenic compositions.

Construction of suitable expression vectors containing desired coding,non-coding, and control sequences employ standard ligation techniques.Isolated plasmids or DNA fragments are cleaved, tailored, and re-ligatedin the form desired to construct the required plasmids. To confirmcorrect sequences in the plasmids constructed, the ligation mixtures canbe used, for example, to transform a host cell and successfultransformants selected by antibiotic resistance where appropriate.Plasmids from the transformants are prepared, analyzed by restrictionand/or sequenced by, for example, by the method disclosed in Messing, etal. (Nucleic Acids Res., 9:309 (1981)), Maxam, et al. (Methods inEnzymology 65:499 (1980)), or other suitable methods which will be knownto those skilled in the art. Size separation of cleaved fragments can beperformed using conventional gel electrophoresis as described, forexample, by Maniatis, et al. (Molecular Cloning, pp. 133-134 (1982)).

Host cells can be transformed with the expression vectors describedherein and cultured in conventional nutrient media modified as isappropriate for inducing promoters, selecting transformants, oramplifying genes. The culture conditions, such as temperature, pH andthe like, are those previously used with the host cell selected forexpression, and will be apparent to the ordinarily skilled artisan.

Steps involved in the purification of one or more of the polypeptides,such as the Trojan antigens, of this invention include (1)solubilization of the desired protein, (2) the development of one ormore isolation and concentration procedures, (3) stabilization of theprotein following purification, and (4) development of a suitable assayto determine the presence of the desired protein. Various aspects ofprotein isolation and purification are discussed in detail in Cooper, T.G., “The Tools of Biochemistry,” John Wiley & Sons, New York, 1977. Asthe techniques of protein isolation and purification are notoriouslywell known in the art, this disclosure will refrain from discussing themin detail. Nevertheless, elements of the cited reference are summarizedand discussed below.

Solubilization is required of most proteins that are to be purified, asmost isolation procedures commonly used operate in aqueous solutions. Insome cases, solubilization can be achieved by merely lysing a host cellwithin which a desired protein has been expressed. In other situations,additional steps, such as extracting the desired protein from asubcellular organelle, may be required. Osmotic lysis, grinding, the useof blenders, ultrasonic waves, presses, and other well known techniquesof protein solubilization can be used with the methods disclosed herein.

There are a variety of techniques available that are well known in theart for the isolation and concentration of the proteins of thisinvention. These techniques include, but are not limited to, (1)differential solubility, (2) ion exchange chromatography, (3) absorptionchromatography, (4) molecular sieve techniques, (5) affinitychromatography, (6) electrophoresis, and (7) electrofocusing. Each ofthese techniques can also be useful in the purification of a protein ofthis invention.

Stabilizing and maintaining a purified protein product in a functionalstate warrants attention to a number of different conditions such as (1)pH, (2) degree of oxidation, (3) heavy metal concentration, (4) mediumpolarity, (5) protease concentration, and (6) temperature. One ofordinary skill in the art would readily know which of the availabletechniques to use to maintain purified protein in an active form withoutundue experimentation.

The Trojan antigens and other polypeptides of the present invention canfurther be prepared using an synthetic peptide synthesizer.

Also included in the present invention are polynucleotide moleculesencoding the Trojan antigens and other polypeptides, including: apolynucleotide molecule encoding amino acids 1-35 of SEQ ID NO:15, apolynucleotide molecule encoding amino acids 1-47 of SEQ ID NO:17, apolynucleotide molecule encoding amino acids 1-21 of SEQ ID NO:19, and apolynucleotide molecule amino acids 1-43 of SEQ ID NO:22.

The present invention further comprises expression vectors comprisingone of the following polynucleotide molecules: a polynucleotide moleculeencoding amino acids 1-35 of SEQ ID NO:15, a polynucleotide moleculeencoding amino acids 1-47 of SEQ ID NO:17, a polynucleotide moleculeencoding amino acids 1-21 of SEQ ID NO:19, and a polynucleotide moleculeamino acids 1-43 of SEQ ID NO:22.

The present invention additionally includes a host cell comprising anexpression vector, wherein the expression vector comprises one of thefollowing polynucleotide molecules: a polynucleotide molecule encodingamino acids 1-35 of SEQ ID NO:15, a polynucleotide molecule encodingamino acids 1-47 of SEQ ID NO:17, a polynucleotide molecule encodingamino acids 1-21 of SEQ ID NO:19, and a polynucleotide molecule aminoacids 1-43 of SEQ ID NO:22.

The present invention further includes methods of preparing apolypeptide, comprising culturing a host cell comprising an expressionvector, which in turn comprises a polynucleotide molecule, underconditions promoting expression of the polypeptide encoded by thepolynucleotide molecule and recovering the polypeptide from the cellculture. In preferred embodiments, the polynucleotides include: apolynucleotide molecule encoding amino acids 1-35 of SEQ ID NO:15, apolynucleotide molecule encoding amino acids 1-47 of SEQ ID NO:17, apolynucleotide molecule encoding amino acids 1-21 of SEQ ID NO:19, and apolynucleotide molecule amino acids 1-43 of SEQ ID NO:22.

Immunogenic Compositions

Included within the present invention are immunogenic compositionscomprising one or more Trojan antigens and a pharmaceutically acceptablecarrier, diluent or adjuvant.

The immunogenic compositions of the present invention may comprise oneTrojan antigen, or two or more different Trojan antigens. Embodiments ofimmunogenic compositions of the present invention include thosecomprising one or more of the specific Trojan antigens discussed herein.

In a preferred example of an immunogenic composition, the compositioncomprises the MAGE-A3 Trojan antigenKVAELVHFLRVKRFLWGPRALVRVKRVIFSKASSSLQLRKKRRQRRR (SEQ ID NO:17) and theHPV 16 E7 Trojan antigen TLGIVXPIRVKRPAGQAEPDRAHYNIVTFXXKXDRKKRRQRRR(SEQ ID NO:22), where each X is independently cysteine or aminobutyricacid, preferably each X is aminobutyric acid, and a pharmaceuticallyacceptable carrier, diluent or adjuvant.

Other preferred embodiments of immunogenic compositions of the presentinvention comprises one or more of the following Trojan antigens: SEQ IDNO:15, SEQ ID NO:17, SEQ ID NO:19, and SEQ ID NO:22, and apharmaceutically acceptable carrier, diluent or adjuvant. In SEQ IDNO:19, X may be cysteine or aminobutyric acid, preferably aminobutyricacid. In SEQ ID NO:22, each X may independently be cysteine oraminobutyric acid, preferably aminobutyric acid.

Preferred examples of pharmaceutically acceptable carriers, diluents andadjuvants include: (1) Dulbecco's phosphate buffered saline, pH ˜7.4,containing about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9%saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose. Other acceptablecarriers, diluents and adjuvants include, but are not limited to saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof, buffers, antioxidants such as ascorbic acid, low molecularweight (less than about 10 residues) polypeptides, proteins, aminoacids, carbohydrates including glucose, sucrose or dextrins, chelatingagents such as EDTA, glutathione and other stabilizers and excipientscommonly employed in pharmaceutical compositions. The composition may beformulated as a lyophilizate using appropriate excipient solutions (e.g.sucrose) as diluents.

In addition to the immunogenic compositions comprising polypeptides,such as the Trojan antigen, included in the present invention areadditional forms of immunogenic compositions. In one embodiment,nucleotide-containing immunogenic compositions are contemplated. Forexample, in one embodiment a Trojan antigen-encoding polynucleotidepreparation including DNA or RNA that encodes an Trojan antigenic may beused for administration to a subject. Nucleotide-containing immunogeniccompositions also include live viral immunogenic compositions. Theviruses for use in the viral immunogenic compositions may includeimmunostimulatory polynucleotides.

Method of Generating an Immune Response

The present invention also includes methods of generating an immuneresponse in a subject. The methods of generating an immune responsegenerally involves administration of a Trojan antigen, or an immunogeniccomposition comprising a Trojan antigen, to a subject.

A preferred embodiment of the present invention is a method ofgenerating an immune response in a subject comprising administering aTrojan antigen comprising amino acids 1-35 of SEQ ID NO:15 to a subjectin an amount sufficient to induce an immune response in said subject.

Another preferred embodiment of the present invention is a method ofgenerating an immune response in a subject comprising administering aTrojan antigen comprising amino acids 1-47 of SEQ ID NO:17 to a subjectin an amount sufficient to induce an immune response in said subject.

A further preferred embodiment of the present invention is a method ofgenerating an immune response in a subject comprising administering aTrojan antigen comprising amino acids 1-21 of SEQ ID NO:19 to a subjectin an amount sufficient to induce an immune response in said subject. InSEQ ID NO:19, X may be cysteine or aminobutyric acid, preferablyaminobutyric acid.

A equally preferred embodiment of the present invention is a method ofgenerating an immune response in a subject comprising administering aTrojan antigen comprising amino acids 1-43 of SEQ ID NO:22 to a subjectin an amount sufficient to induce an immune response in said subject. InSEQ ID NO:22, each X may independently be cysteine or aminobutyric acid,preferably aminobutyric acid.

A further preferred embodiment of the present invention is a method ofgenerating an immune response in a subject comprising administering a(a) Trojan antigen comprising amino acids 1-47 of SEQ ID NO:17 and (b) aTrojan antigen comprising amino acids 1-43 of SEQ ID NO:22 to a subjectin an amount sufficient to induce an immune response in said subject. InSEQ ID NO:22, each X may independently be cysteine or aminobutyric acid,preferably aminobutyric acid.

In each of these embodiments, the Trojan antigens may co-administeredwith a pharmaceutically acceptable carrier, diluent or adjuvant, in theform of an immunogenic composition.

Preferred examples of pharmaceutically acceptable carriers, diluents andadjuvants include: (1) Dulbecco's phosphate buffered saline, pH ˜7.4,containing about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9%saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose. Other acceptablecarriers, diluents and adjuvants include, but are not limited to saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof, buffers, antioxidants such as ascorbic acid, low molecularweight (less than about 10 residues) polypeptides, proteins, aminoacids, carbohydrates including glucose, sucrose or dextrins, chelatingagents such as EDTA, glutathione and other stabilizers and excipientscommonly employed in pharmaceutical compositions. The composition may beformulated as a lyophilizate using appropriate excipient solutions (e.g.sucrose) as diluents.

The Trojan antigens may be administered (alone or in the context of animmunogenic composition) in a dosage containing between about 5 ug andabout 100 mg of peptide, more preferably in a dosage containing betweenabout 300 ug and about 1 mg of peptide. More preferred dosages are 300ug, 350 ug, 400 ug, 450 ug, 500 ug, 550 ug, 600 ug, 650 ug, 700 ug, 750ug, 800 ug, 850 ug, 900 ug, 950 ug and 1 mg of peptide.

The methods of generating an immune response as disclosed herein mayalso include the administration of additional compounds to augment thegeneration of an immune response. For example, in addition to the Trojanantigen and immunogenic compositions comprising a Trojan antigen,additional compounds may be administered before or after the Trojanantigen or immunogenic composition, or co-administered with the Trojanantigen or immunogenic composition.

Such additional compounds include Montanide and GM-CSF. Montanide(Montanide ISA-51) is a human-approved adjuvant, similar to incompleteFreunds adjuvant, recognized to regulate the temporal aspects of peptiderelease (Aichele et al. T cell priming versus T cell tolerance inducedby synthetic peptides. Journal of Experimental Medicine 182(1):261-6(1995)), used in human vaccine therapy to stimulate the immune system.GM-CSF (granulocyte macrophage-colony stimulating factor) is a cytokineinvolved in the growth and differentiation of myeloid and monocyticlineage cells, including dendritic cells, monocytes and tissuemacrophages and cells of the granulocyte lineage.

Additional compounds include adjuvants of the Toll-like receptor family.These adjuvants include CpG-containing oligodeoxynucleotides, bacterialDNA, polyinosinic-polycytidylic acid, synthetic double-stranded RNA,synthetic IMIQUOMOD™, and RNA of viral or bacterial origin, or a viralRNA mimic.

In a preferred embodiment, both Montanide and GM-CSF are co-administeredto a subject with the immunogenic composition. Montanide may beadministered at a dosage of between about 0.1 mL and 10 mL, preferablyat between 0.25 mL and 2 mL, more preferably at 1.2 mL GM-CSF may beadministered at a dosage of between about 5 ug/m² and 1 mg/m²,preferably at between 20 ug/m² and 500 ug/m², more preferably at 100ug/m².

Methods of Treatment

Also included in the present invention are methods of treating a subjecthaving SCCHN. The methods of treatment generally involved administrationof a Trojan antigen, or an immunogenic composition comprising a Trojanantigen, to a subject having SCCHN.

A preferred embodiment of the present invention is a method of treatingSCCHN comprising administering to a subject in need of such treatment atherapeutically-effective amount of a Trojan antigen comprising aminoacids 1-35 of SEQ ID NO:15.

Another preferred embodiment of the present invention is a method oftreating SCCHN comprising administering to a subject in need of suchtreatment a therapeutically-effective amount of a Trojan antigencomprising amino acids 1-47 of SEQ ID NO:17.

A further preferred embodiment of the present invention is a method oftreating SCCHN comprising administering to a subject in need of suchtreatment a therapeutically-effective amount of a Trojan antigencomprising amino acids 1-21 of SEQ ID NO:19. In SEQ ID NO:19, X may becysteine or aminobutyric acid, preferably aminobutyric acid.

A equally preferred embodiment of the present invention is a method oftreating SCCHN comprising administering to a subject in need of suchtreatment a therapeutically-effective amount of a Trojan antigencomprising amino acids 1-43 of SEQ ID NO:22. In SEQ ID NO:22, each X mayindependently be cysteine or aminobutyric acid, preferably aminobutyricacid.

A further preferred embodiment of the present invention is a method oftreating SCCHN comprising administering to a subject in need of suchtreatment a therapeutically-effective amount of (a) a Trojan antigencomprising amino acids 1-47 of SEQ ID NO:17 and (b) a Trojan antigencomprising amino acids 1-43 of SEQ ID NO:22. In SEQ ID NO:22, each X mayindependently be cysteine or aminobutyric acid, preferably aminobutyricacid.

In each of these embodiments, the Trojan antigen may be co-administeredwith a pharmaceutically acceptable carrier, diluent or adjuvant, in theform of an immunogenic composition.

Preferred examples of pharmaceutically acceptable carriers, diluents andadjuvants include: (1) Dulbecco's phosphate buffered saline, pH ˜7.4,containing about 1 mg/ml to 25 mg/ml human serum albumin, (2) 0.9%saline (0.9% w/v NaCl), and (3) 5% (w/v) dextrose. Other acceptablecarriers, diluents and adjuvants include, but are not limited to saline,buffered saline, dextrose, water, glycerol, ethanol, and combinationsthereof, buffers, antioxidants such as ascorbic acid, low molecularweight (less than about 10 residues) polypeptides, proteins, aminoacids, carbohydrates including glucose, sucrose or dextrins, chelatingagents such as EDTA, glutathione and other stabilizers and excipientscommonly employed in pharmaceutical compositions. The composition may beformulated as a lyophilizate using appropriate excipient solutions (e.g.sucrose) as diluents.

The Trojan antigens may be administered (alone or in the context of animmunogenic composition) in a dosage containing between about 5 ug andabout 100 mg of peptide, more preferably in a dosage containing betweenabout 300 ug and about 1 mg of peptide. A preferred dosage is between300 ug and 1 mg. More preferred dosages are 300 ug, 350 ug, 400 ug, 450ug, 500 ug, 550 ug, 600 ug, 650 ug, 700 ug, 750 ug, 800 ug, 850 ug, 900ug, 950 ug and 1 mg of peptide. A physician may determine the actualdosage that will be most suitable for a subject, which may vary with theage, weight and response of the particular subject. The above dosagesare exemplary of the average case. There can, of course, be individualinstances where higher or lower dosage ranges are merited, and such arewithin the scope of this invention.

The methods of generating an immune response as disclosed herein mayalso include the administration of additional compounds to augment thegeneration of an immune response. For example, in addition to the Trojanantigen and immunogenic compositions comprising a Trojan antigen,additional compounds may be administered before or after the Trojanantigen or immunogenic composition, or co-administered with the Trojanantigen or immunogenic composition.

Such additional compounds include Montanide and GM-CSF. Montanide(Montanide ISA-51) is a human-approved adjuvant, similar to incompleteFreunds adjuvant, recognized to regulate the temporal aspects of peptiderelease (Aichele et al. T cell priming versus T cell tolerance inducedby synthetic peptides. Journal of Experimental Medicine 182(1):261-6(1995)), used in human vaccine therapy to stimulate the immune system.GM-CSF (granulocyte macrophage-colony stimulating factor) is a cytokineinvolved in the growth and differentiation of myeloid and monocyticlineage cells, including dendritic cells, monocytes and tissuemacrophages and cells of the granulocyte lineage. Additional compoundsinclude adjuvants of the Toll-like receptor family. These adjuvantsinclude CpG-containing oligodeoxynucleotides, bacterial DNA,polyinosinic-polycytidylic acid, synthetic double-stranded RNA,synthetic IMIQUOMOD™, and RNA of viral or bacterial origin, or a viralRNA mimic.

In a preferred embodiment, both Montanide and GM-CSF are co-administeredto a subject with the immunogenic composition. Montanide may beadministered at a dosage of between about 0.1 mL and 10 mL, preferablyat between 0.25 mL and 2 mL, more preferably at 1.2 mL. GM-CSF may beadministered at a dosage of between about 5 ug/m² and 1 mg/m²,preferably at between 20 ug/m² and 500 ug/m², more preferably at 100ug/m².

In each of the methods described herein, the Trojan antigens andimmunogenic compositions comprising the Trojan antigens may beadministered to a subject by any effective, convenient manner including,for instance, administration by topical, oral, anal, vaginal,intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal orintradermal routes among others. Formulations suitable for parenteraladministration include aqueous and non-aqueous sterile injectionsolutions presented in unit-dose or multi-dose containers. It should bealso understood that, in addition to the ingredients mentioned above,formulations of this invention might include other agents conventionalin the art having regard to the type of formulation in question.

In each of the methods described herein, the Trojan antigens andimmunogenic compositions comprising the Trojan antigens may beadministered to a subject as a one-time dose, or as a series of two ormore doses over prolonged periods of time. In a preferred embodiment,the Trojan antigens and immunogenic compositions of the presentinvention are administered as a series of four doses, with one doseadministered each month for four months. In this embodiment, up to fouradditional doses can be administered over a further four month period.Other preferred dosing schedules include administration daily, once aweek, twice a week, 3-4 times per week, weekly, twice a month and threetimes per month. The number of doses administered varies depending onthe dosing schedule, but dosing can continue under one of the dosingschedules above for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 months.

Appropriate doses for each can readily be determined by techniques wellknown to those of ordinary skill in the art. Such a determination willbe based, in part, on the tolerability and efficacy of a particular doseusing techniques similar to those used to determine proper vaccinedoses. The skilled artisan will understand that combinations of thedosing schedules indicated above can also be used.

Each of the methods described herein may be practiced in vitro, in vivo,or ex vivo.

In addition to the methods for generating an immune response and methodsfor the treatment of SCCHN described herein, the following additionalmethods are included in the present invention. In one embodiment, anucleotide sequence encoding a Trojan antigen is introduced into anexogenous organism using standard molecular biology techniques wellknown to those of ordinary skill in the art, such as through the use ofan expression vector described herein. Exemplary molecular biologytechniques are discussed in Ausubel, et al., “Short Protocols inMolecular Biology.” The resulting recombinant organism can then be usedas an immunogen composition in the methods described herein. In apreferred embodiment, an attenuated pathogenic organism serves as theexogenous organism.

The methods of the present invention may also be practiced using anucleotide-containing immunogenic composition. For example, in oneembodiment, an immune response may be generated in a subject, or asubject may be treated, by administering an Trojan antigen-encodingpolynucleotide preparation including DNA or RNA that encodes an Trojanantigenic to the host. Preferably, the polynucleotide preparation isadministered to a mucosal inductor site in the mucosal tissue of thehost. Naked DNA may be administered directly to the mucosa (e.g., insaline drops) or in a recombinant gene expression vector.

Nucleotide-containing immunogenic compositions also include live viralimmunogenic compositions. The viruses for use in the viral immunogeniccompositions include immunostimulatory polynucleotides. Preferably, aTrojan antigen is administered through its expression by a recombinantgene expression vector.

U.S. Pat. No. 6,110,898, to Malone, et al., entitled, “DNA vaccines foreliciting a mucosal immune response” provides detailed teaching for thegeneration of such immunogenic compositions. In particular, Maloneteaches obtaining a recombinant alphavirus vector system as described inMalone, J. G., et al., “Mucosal immune responses associated withpolynucleotide vaccination”, Behring Inst Mitt 98:63-72 (1997 February).DNA encoding a Trojan antigen (for example) is substituted for the lacZgene in the vector.

Alternatively, one or more polynucleotides encoding a Trojan antigen canbe introduced to an attenuated EAEC, Salmonella spp., Shigella spp.,Lactobacillus spp., or other attenuated bacteria which is invasive formucosal tissue, which then expresses the particular Trojan antigenencoded by the polynucleotide. The bacteria is administered to an animalto generate an immune response to the particular Trojan antigen encoded.

Pre-Screening

Preferably, the methods of the present invention are practiced on asubject that has a SCCHN tumor that expresses (a) HLA-A2 antigens and(b) either MAGE-A3 or HPV 16 E7 proteins, or (c) HLA-A2 antigens andboth MAGE-A3 and HPV 16 E7 proteins.

A subject may first be typed to determine whether they are HLA-A2positive. A subject can be typed, for example, by obtaining a bloodsample, followed by PBL typing using a commercially available HLA-Adetection kit (Dynal) according to manufacturer's instructions. PBLs maybe also typed by PCR or flow cytometric analysis (using, for example,BB7.2 mouse anti-human HLA-A2 mAb, available from the ATCC), with knownpositive and negative controls (Hoffmann et al. Frequencies of Tetramer+T Cells Specific for the Wild-Type Sequence p53264-272 Peptide in theCirculation of Patients with Head and Neck Cancer. Cancer Research62(12):3521-3529 (2002)).

Tumors from patients who are HLA-A2 positive may then be evaluated forthe expression of HLA-A2, MAGE-A3, and HPV16 E7. HLA-A2 expression maybe evaluated by immunohistochemistry. For example, following biopsytumors can be frozen in OCT blocks, sectioned onto glass slides, andstained with hematoxylin and eosin (H&E), anti-HLA-A2 antibody andsecondary antibody, or secondary antibody alone per the DAKO EnVision+Protocol (DAKO Corporation, Carpinteria, Calif.). Normal lung parenchymafrom a known HLA-A2 positive patient may serve as a positive control.

HPV 16 E7 and MAGE-A3 detection may be performed using techniquespreviously published (Strome et al. Squamous Cell Carcinoma of theTonsils: A Molecular Analysis of HPV Associations. Clinical CancerResearch 18:1093-1100 (2002); Kienstra et al. Identification ofNY-ESO-1, MAGE-1, and MAGE-3 in head and neck squamous cell carcinoma.Head and Neck 25(6):457-463 (2003)). Briefly, samples may be amplifiedusing specific primers to:

E6 region of HPV 16 (325 bp) (SEQ ID NO: 23)5′-CCACAGTTATGCACAGAGCTGCAAACAACTATACAT (HPV 16-E6-140-36D) (SEQ ID NO:24) 5′-TTGTCCAGATGTCTTTGCTTTTCTTCAGGACACAGT (HPV16-E6-465-36U) MAGE-A3primer (423 bp) (SEQ ID NO: 25) 5′-GAAGCCGGCCCAGGCTCG (SEQ ID NO: 26)5′-GGAGTCCTCATAGGATTGGCTCC

The amplification reaction may be performed in 50 μl containing 10 mMTris pH 8.3, 50 mM KCl, 2.0-mM MgCl2, 200 μM each dNTP (100 μM dUTP and100 μM dTTP), 2.5 units Amplitag gold (5 U/μl, Perkin Elmer), 0.1%bovine serum albumin (BSA), 19.5 μl RNase free water, 0.5 μm of eachprimer and 5 μl of sample DNA. PCR cycling conditions may be 95° C. for10 min followed by 40 cycles of 95° C. for 1 min, 55° C. for 1 min, 72°C. for 1 min followed by 10 min at 72° C. After amplification, 15 μl ofeach sample may be run on a 2% agarose gel containing 20 μg ethidiumbromide in an 100 ml gel to visualize products. DNA from the Caski cellline and DNA from a known MAGE-A3 positive tumor may be used as apositive PCR control to assess the success of the amplification. PCRreagents lacking DNA (no sample added) may serve as a negative controlin each PCR amplification.

EXAMPLES Example 1 Identification of MAGE-A3 Antigenic Epitopes

Using a predictive algorithm based on the presence of MHC bindingmotifs, an HLA-A1-binding peptide from MAGE-A3 that induced in vitroanti-tumor CTL responses with lymphocytes from normal individuals wasidentified (Celis et al. Induction of anti-tumor cytotoxic T lymphocytesin normal humans using primary cultures and synthetic peptide epitopes,PNAS USA 91(6):2105-2109 (1994)). In addition to the HLA-A1-restrictedepitope, an HLA-A2 restricted CTL epitope, which is more frequentlyfound in the general population than HLA-A1, was identified (Kawashimaet al. The multi-epitope approach for immunotherapy for cancer:identification of several CTL epitopes from various tumor-associatedantigens expressed on solid epithelial tumors. Human Immunology59(1):1-14 (1998)). CTL-induced by peptide MAGE-A3₁₁₂₋₁₂₀ (KVAELVHFLL;SEQ ID NO:2) were quite effective in recognizing tumor cells expressingMAGE-A3 antigen and HLA-A2 (FIG. 1).

Boon et al. reported the existence of another HLA-A2-restricted epitopefrom MAGE-A3, namely MAGE-A3₂₇₁₋₂₇₉ (FLWGPRALV; SEQ ID NO:1). Thisepitope was also efficient in inducing CTL responses to tumorsexpressing MAGE-A3 antigen (van der Bruggen et al. A peptide encoded byhuman gene MAGE-3 and presented by HLA-A2 induces cytolytic Tlymphocytes that recognize tumor cells expressing MAGE-3. EuropeanJournal of Immunology 24(12):3038-43 (1994)).

In summary, the two HLA-A2-restricted CTL epitopes described above,MAGE-A3₁₁₂₋₁₂₀ and MAGE-A3₂₇₁₋₂₇₉, have been proven to be effective ingenerating cytotoxic responses against tumors expressing the MAGE-A3antigen.

Example 2 Identification of a Promiscuous T Helper Epitope from MAGE-A3

A promiscuous T helper epitope that is presented to T cells by HLA-DR4and HLA-DR7, two of the most frequently found MHC class II alleles, wasidentified. Peptide MAGE-A3₁₄₉₋₁₆₀ (VIFSKASSSLQL; SEQ ID NO:5) was foundto stimulate T helper lymphocytes that recognized recombinant MAGE-A3protein or cell lysates from tumors expressing MAGE-A3 antigen(Kobayashi et al. Tumor-reactive T helper lymphocytes recognize apromiscuous MAGE-A3 epitope presented by various majorhistocompatibility complex class II alleles. Cancer Research61(12):4773-8 (2001)). As shown in FIG. 2, HLA-DR4-restricted HTL clone8G9 recognizes naturally processed MAGE-A3 antigen. A: ProliferativeT-cell response induced by MAGE-A3₁₄₆₋₁₆₀ (+Peptide), recombinantMAGE-A3 protein (rMAGE-A3) or recombinant gp100 (rgp100). B: Tissueculture supernatants from experiment described in panel A, werecollected after 48 hr and the concentration of GM-CSF was measured byELISA. C: T-cell clone 8G9 recognizes UV-irradiated melanoma cells thatexpress MAGE-A3 (HT-144 (+), SKmel-28 (+), 697mel (+)) via antigencross-presentation by autologous DC. DC incubated with MAGE-A3 negativemelanoma cell line (888mel (−)) did not stimulate the T-cell clone.Autologous DC were incubated with irradiated melanoma cells at a 1:1ratio for 48 hours. The antigen-pulsed DC were then mixed with HTL (at a1:20 ratio) and 2 days later culture supernatants were collected andassayed for the presence of GM-CSF. Values shown are the means oftriplicate determinations; bars, SD.

Example 3 Identification and Selection of CTL and T Helper Epitopes fromHPV 16 E7

Two HLA-A2-restricted CTL epitopes and one T helper epitope wereselected. The identification and description of these epitopes have beenpublished (de Jong et al. Frequent detection of human papillomavirus 16E2-specific T-helper immunity in healthy subjects. Cancer Research62(2):472-479 (2002); Kast et al. Role of HLA-A motifs in identificationof potential CTL epitopes in human papillomavirus type 16 E6 and E7proteins. Journal of Immunology 152(8):3904-3912 (1994)). Furthermore,the HLA-A2-restricted CTL epitopes were shown to induce CTL responses(Jager et al. Monitoring CD8 T cell responses to NY-ESO-1: correlationof humoral and cellular immune responses. PNAS USA 97(9):4760-4765(2000); den Haan et al. Identification of a graft versus hostdisease-associated human minor histocompatibility antigen. Science268(5216):1476-1480 (1995); Bennouna et al. Application of IL-5 ELISPOTassays to quantification of antigen-specific T helper responses. Journalof Immunological Methods 261(1-2):145-156 (2002)).

Example 4 Control Experiment Using Ovalbumin Antigenic Epitope

A Trojan antigen comprising the mouse CTL epitope from ovalbumin(SIINFEKL; SEQ ID NO:11), coupled to the HIV TAT transporter peptide(RKKRRQRRR; SEQ ID NO:9) via an AAA linked was preparing, yielding theRKKRRQRRRAAASIINFEKL (SEQ ID NO:12).

The capacity of the ovalbumin Trojan antigen to be processed andpresented by DC to OT-1 T cells specific for the SIINFEKL (SEQ ID NO:11)epitope was studied. As shown in FIG. 3, DC that were pulsed with theovalbumin Trojan antigen remained stimulatory for T cells after a 48hour period of incubation, while the DC that were incubated with theantigenic epitope alone had lost most of their stimulatory activity.This experiment demonstrated that pulsing the DC with Trojan antigenallowed these APC to present the epitope for a longer period of time,compared with APC pulsed with only the SIINFEKL (SEQ ID NO:11) antigenicepitope. C57/BL6 DC were pulsed with either SIINFEKL or TrojAg(RKKRRQRRRRAAASIINFEKL) for 2 hours, washed three times, and then keptin 37° C. After 48 hours, the ability of peptide-loaded DC to induceIFN-gamma release from OT-1 T cells was determined by ELISA. Theconcentration of IFN-gamma was determined at various dilutions of thesupernatant that was collected after 24 hours of incubation of the DCwith the OT-1 T cells.

Example 5 HPV 16 E7 Trojan Antigens Stimulate Interferon Gamma Releasefrom HLA-A2 T Cells

To study the ability of the HPV 16 E7 Trojan antigen to stimulatefunctional T cell reactivity, levels of Interferon Gamma (IFN γ) releasefrom Trojan antigen-stimulated HLA-A2 positive T cells versus IFN γproduction from T cells stimulated with the constituent peptideepitopes, were determined. Briefly, freshly isolated HLA-A2 positiveCD8+ (CTLs) and CD4+ (HTLs) were positively selected by Dynal magneticbead separation (Dynal ASA). T cells were stimulated with irradiatedautologous CD8−/CD4− PBLs (flow-through from the magnetic beadseparation) pulsed with TLGIVXPIRVKRPAGQAEPDRAHYNIVTFXXKXDRKKRRQRRR (SEQID NO:22) or the constituent HLA-A2.1 epitope TLGIVXPI (SEQ ID NO:18),where each X is aminobutyric acid, for 48 hours in 96-well Immobilon-Pmembrane multiscreen plate (Millipore) coated with IFN γ-specificcapture antibody (BD Biosciences for IFN γ). The wells were washed,treated with Biotin-conjugated detection antibody and3-amino-9-ethylcarbazole (AEC) for substrate development.

Spots were counted by first obtaining digitized images of the wells(performed by C.T.L. Analyzers, Cleveland, Ohio) and then analyzingthese images with software purchased from C.T.L. Analyzers. Thefrequency of spots (250,000 cells per well) obtained with the Trojan andconstituent peptides was compared and found to be equivalent at 0.02% Tcell reactivity for both peptides. The results of this experiment areshown in FIG. 4.

Example 6 Expression Patterns of HLA-A2 in SCCHN

To demonstrated the ability to quantitate HLA-A2 expression on freshSCCHN, immunohistochemical analysis was performed on five freshlyisolated SCCHN, using the HLA-A2 specific mAb SB03-111 (kindly providedby Dr. Soldano Ferrone, Roswell Park Cancer Institute, Buffalo, N.Y.).

As shown in FIG. 5, stromal tissue demonstrated a membranouscytoplasmic-staining pattern. Tissue from patients who were negative forHLA-A2 by PCR, did not display immunoreactivity to this mAb. In oneHLA-A2 positive patient, the parenchyma was observed to express HLA-A2,but the tumor was negative.

The samples were prepared using freshly isolated tumors snap frozen inOCT. 5 micron sections were cut onto charged glass slides. Specimenswere stained with H&E, mouse Ig control or SB03-111, using previouslypublished techniques (Dong et al. Tumor-associated B7-H1 promotes T-cellapoptosis. A potential mechanism of immune evasion. Nature Medicine8:793-800 (2002)). The optimal dilution for SB03-111 was 1/2500 (datanot shown).

All documents and publications referenced herein are hereby expresslyincorporated by reference in their entirety. In particular, GrantApplication No. DE015324 is hereby expressly incorporated by referencein its entirety.

The invention of this application has been described above bothgenerically and with regard to specific embodiments. Although theinvention has been set forth in what is believed to be the preferredembodiments, a wide variety of alternatives known to those of skill inthe art can be selected within the generic disclosure. The invention isnot otherwise limited, except for the recitation of the claims set forthbelow.

1. (canceled)
 2. A isolated polypeptide comprising amino acids 1-47 ofSEQ ID NO:
 17. 3-6. (canceled)
 7. An immunologic composition comprisinga polypeptide of claim 2, and a pharmaceutically acceptable carrier,diluent or adjuvant.
 8. (canceled)
 9. A method of generating an immuneresponse in a subject comprising administering a polypeptide comprisingamino acids 1-47 of SEQ ID NO: 17 to a subject in an amount sufficientto induce an immune response in a subject.
 10. (canceled)
 11. (canceled)12. The method of claim 9, wherein said polypeptide is administered inconjunction with a pharmaceutically acceptable carrier, diluent oradjuvant.
 13. (canceled)
 14. The method of claim 9, wherein saidpolypeptide is administered in an amount of between 100 μg and 1.5 mg.15. (canceled)
 16. The method of claim 9, wherein said polypeptides areadministered in a combined amount of about 1 mg.
 17. (canceled)
 18. Themethod of claim 9, wherein said polypeptide is co-administered withmontanide, in an amount of between 0.5 and 1.5 mL, and GM-CSF, in anamount of between 50 and 150 μg/m².
 19. (canceled)
 20. A method oftreating squamous cell carcinoma of the head and neck (SCCHN) comprisingadministering to a subject in need of such treatment atherapeutically-effective amount of a polypeptide comprising amino acids1-47 of SEQ ID NO:17.
 21. (canceled)
 22. (canceled)
 23. The method ofclaim 20, wherein said polypeptide is co-administered with apharmaceutically acceptable carrier, diluent or adjuvant.
 24. (canceled)25. The method of claim 20, wherein said polypeptide is administered inan amount between 100 μg and about 1.5 mg.
 26. (canceled)
 27. The methodof claim 20, wherein said polypeptide is administered in an amount ofabout 1 mg.
 28. (canceled)
 29. The method of claim 20, wherein saidpolypeptide is co-administered with montanide, in an amount of betweenabout 0.5 and 1.5 mL, and GM-CSF, in an amount of between 50 and 150μg/m². 30-42. (canceled)